Nanosystems for controlled transport of active molecules for diagnostic, prognostic and therapeutic purposes

ABSTRACT

Disclosed is a nanoparticle system consisting of a polymer support or substrate in the form of nanoparticles to which a hydrolase enzyme able to degrade hyaluronic acid and one or more biologically and/or pharmacologically active molecules are covalently bonded, its preparation process and its uses in the diagnostic, prognostic and therapeutic fields.

The invention relates to a nanoparticle system consisting of a polymersupport or substrate in the form of nanoparticles to which a hydrolaseenzyme able to degrade hyaluronic acid and one or more biologicallyand/or pharmacologically active molecules are covalently bonded, itspreparation process and its uses in the diagnostic, prognostic andtherapeutic fields.

PRIOR ART

The use of nanoparticles to transport medicaments through thecirculatory system is a known technique, which is becoming increasinglywidely used due to major technological progress in the field. It offersconsiderable advantages over conventional administration methods; wheninjected into the bloodstream, nanoparticles, propelled by haemodynamicactions, reach the site of the pathological process and, after adheringto the walls of the “sick” cell, release the medicament locally at therequired dose; they consequently have a highly selective action.Nanoparticles are particles with nanometric dimensions, which usuallyconsist of a carrying structure that contains the medicament or, moregenerally, an active molecule; nanoparticles should be designed in sucha way that regardless of the chosen administration route, the release ofthe medicament associated with them is calibrated in both qualitativeand quantitative terms to provide:

a) high selectivity, only detecting and acting against sick cells, or atthe target site in general;

b) high efficacy, so that the minimum effective amount of the activemolecule is used;

c) low toxicity, as a direct result of point b).

The studies conducted to date to evaluate nanoparticle systemsdemonstrate that they represent, in vitro, an effective administrationroute for medicaments and/or active molecules; in vivo, however,consideration must be given to the ability of the nanoparticles toovercome the barriers inherent in each administration route, thedistribution volume required to achieve the therapeutic effect, andabove all the toxicity which may result; the size, type and compositionof said particles must therefore be precisely calibrated in relation tothe intended applications. For the most common administration routes(oral, injection), the most critical factors are associated with thefact that the nanoparticles may release too much or too little of theactive ingredient, becoming toxic in the first case and useless in thesecond; moreover, after oral administration, the hepatic first-passeffect can significantly affect the blood concentration needed for thepharmacological activity.

The need to control more directly and optimize the concentration of themedicament in the blood has led to the development of alternativeadministration systems through the skin (transdermal route) or themucosa (transmucosal route, which comprises the sublingual/buccal,nasal, tracheal and rectal routes). Said systems enable the medicamentto be introduced rapidly into the bloodstream, eliminating the risks anddifficulties described above, since both skin and mucosa have a highdensity of blood vessels which allow rapid systemic diffusion of themedicament. Even in these cases, however, the natural barrier that thenanoparticles must cross must be considered; for example, thetranscutaneous route requires the nanoparticles to penetrate the skin,and this problem has been dealt with to date by co-administration of themedicament with enzymes that promote said penetration, such ashyaluronidase. Hyaluronidase hydrolyses the hyaluronic acid present inthe tissues, depolymerizing the viscoelastic structure of thesubcutaneous interstitial matrix and thus increasing the dispersion ofthe locally injected nanoparticles (Bookbinder et al., J Contr Rel 2006,28, 230-241). Some technical solutions that combine an active ingredientwith hyaluronidase and with free polymers or polymers organized intomicro- or nanoparticles are already known; however, said solutions arecharacterised in that the various ingredients are mixed together, or atmost, the nanoparticles incorporate active ingredients and/or the enzymehyaluronidase, which are inserted into the particles during theformation process (WO2013151774; U.S. 2013302400; Rajan et al., Int JPharmac, 2013, 453, 514-522). However, with this process, theingredients as a whole obviously cannot be precisely measured in orderto achieve a constant, continuous release of active ingredient at themaximum therapeutic doses and with the minimum toxicity. In any event,said solutions do not ensure that the medicament and particle remainbonded until they reach their target site.

DESCRIPTION OF THE INVENTION

The present invention overcomes the current problems by using alreadystructured nanoparticles which are bonded with covalent bonds to one ormore active substances, in quantities calculated to ensure that theyperform the desired function, and the enzyme hyaluronidase, which hasthe function of promoting penetration.

DESCRIPTION OF FIGURES

FIG. 1. Schematic structure of the nanosystem according to the inventionconsisting of a core polymer (nanoparticle) bonded to a branched carbonchain containing one or more groups suitable for covalent bonding ofactive molecules.

FIG. 2. Sample schema for covalent coupling of rHyal_sk to 200 nmpolystyrene nanoparticles.

FIG. 3. Graph showing results of the enzymatic activity of rHyal_skcovalently bonded to nanoparticles (turbidimetric assay). The graphindicates the number of HYAL units coupled to the nanoparticles.

FIG. 4. Scheme portraying covalent bifunctionalization of nanoparticleswith rHyal_Sk and fluorophore or medicament.

FIG. 5. Scheme portraying covalent bifunctionalization of nanoparticleswith rHyal_Sk and antibody (Ab).

FIG. 5B. Scheme showing covalent bifunctionalization of nanoparticleswith rHyal_Sk and antibody (Ab) and preparation fortrifunctionalization.

FIG. 5C. Scheme showing covalent trifunctionalization of nanoparticleswith fluorophore, rHyal_Sk and antibody (Ab).

FIG. 6. Validation of nanosystems labelled with Cy5 and Cy7. a) Generalchemical structure; b) flow cytofluorometry data of nanosystems labelledwith Cy5 and Cy7.

FIG. 7. Graph showing results of enzymatic activity of nanosystemsbifunctionalised with rHyal_Sk and fluorophores (Cy5 and Cy7)(turbidimetric assay).

FIG. 8. Flow cytofluorometry data of cell penetration by nanosystemsobtained as described in Example 2 on (A) HUVEC and (B) HFF cell lines.

FIG. 9. A) Count of number of Cy5-HyaluSpheres and Cy5-FluoroSpherespresent in the lower part of the insert (Transwell). B) In vitroTranswell model with two HUVEC monolayers. C) Fluorescence microscopy ofcell penetration by Cy5-HyaluSpheres of the HUVEC monolayer in the lowerpart of the insert.

FIG. 10. Cytotoxic activity of doxorubicin covalently bonded tonanoparticles on cell line 4T1.

FIG. 11. Cytotoxic activity of doxorubicin covalently bonded tonanoparticles on cell line A549.

FIG. 12. Validation of conjugation to monoclonal antibody on nanosystemsobtained as described in Example 4B (agarose gel electrophoresis test).

FIG. 13. Validation of conjugation to monoclonal antibody on nanosystemsobtained as described in Example 4C (agarose gel electrophoresis test).

FIG. 14. Flow cytofluorometry data of cell penetration by nanosystemsobtained as described in Example 4C on A549 and H520 cell lines.

DETAILED DESCRIPTION OF THE INVENTION

The nanoparticle system according to the invention consists of a polymersupport or substrate in the form of nanoparticles to which a hydrolaseenzyme able to degrade hyaluronic acid (hyaluronidase) and one or morebiologically and/or pharmacologically active molecules are covalentlybonded, its preparation process and its uses in the diagnostic,prognostic and therapeutic fields.

The nanoparticles according to the invention, due to the type of bondwith which they are anchored to the target molecules, represent aninnovative system for the transport and controlled, calibrated releaseof active ingredients, which simultaneously guarantees high selectivity,high efficacy and low toxicity.

Unlike the systems currently known, all the ingredients of thenanoparticle system according to the invention are combined by means ofstable covalent bonds to form a single nanosystem with hyaluronidaseactivity able to bind and transport other functionally active molecules;they are therefore no longer mechanically separable from one another,and operate as a single unit. The nanosystem described herein consistsof a polymer core (nanoparticle) bonded to a branched heterocarbon chaincontaining one or more groups able to bond the target activeconstituents, in order to form a single active molecular entity, thestructure of which is schematically illustrated in FIG. 1.

Polymer Core

The polymer core in the form of a nanobead consists of natural orsynthetic polymers or copolymers comprising functional groups able toreact with functional groups of the heterocarbon chain. Examples ofpolymers include polystyrene (PS) functionalised with amino groups,polylactic acid (PLA), polylactic coglycolic acid (PLGA),poly(N-vinylpyrrolidone) (PVP), polyethylene glycol (PEG),polycaprolactone, polyacrylic acid (PAA), polymethyl methacrylate andpolyacrylamide. Examples of natural polymers which can be used toprepare the nanoparticles include chitosan, gelatin, sodium alginate andalbumin. Particularly suitable for the purposes of the invention ispolystyrene functionalised with amino groups, which is obtainable, forexample, by a process of polymerization in dispersion (Sanchez-Martin etal, 2005, ChemBioChem, 6,1341-1345) from aqueous solutions of: a)styrene as main monomer; b) divinylbenzene (DVB), a crosslinking agentcontaining two vinyl groups; c) vinylbenzylamine hydrochloride (VBAH) assecond monomer containing an amino group. The reaction is conducted at80° C. in an inert atmosphere with magnesium sulphate as stabilisingagent. A water-soluble radical initiator,2-dihydrochloride-2″-azobis-(2-methylpropionamide) (V-50), is added 30minutes after the start of the reaction. The reaction continues for afurther 2 hours under stirring. Nanoparticles of dimensions rangingbetween 50 and 2000 nanometres are obtained in this way. Polystyrenenanoparticles of dimensions ranging between 100 and 500 nm, and inparticular amounting to 200 nm, are preferred. The synthesis describedwas optimized to obtain 200 nm nanoparticles functionalised with aminogroups. The dimensions were checked and the nanoparticles dispersed withlaser diffractometry [Dynamic Light Scattering (DLS) Zetasizer Nano ZS,Malvern Instruments] and scanning electron microscopy (SEM, Hitachi,S-510).

Heterocarbon Chain

The branched heterocarbon chain (hereinafter called “carbon chain”)contains one or more groups suitable to form a covalent bond with activemolecules and simultaneously generates a space between the nanoparticleand said molecules. Said chains are variable-length chains having groupsable to form covalent bonds, in particular groups orthogonal to oneanother, which are deprotected by various procedures during synthesis.Their presence improves biocompatibility and reduces the interactionsbetween the nanoparticle and the active molecules, thus increasing thestability and functionality of the latter. Examples of said chainsinclude chains saturated with a number of carbons ranging between 6 and24; methoxypolyethylene glycol chains; dimethyl suberimidate chain;chains of polyalkyl glycols, in particular polyethylene glycols, amongwhich the N-Fmoc-N-succinyl-4,7,10-trioxa-1,13-tridecanediamine chain(PEG spacer) is particularly preferred. The length of the chain can alsobe modified as required (steric bulk of the molecules to be bonded,reaction conditions, etc.) by adding spacers; polyethylene glycol (PEG)chains are generally used for this purpose.

Substituents: Group X

Group X (FIG. 1) represents the enzymatic component (hyaluronidase),which may be of human or animal origin, from vertebrates, bacteria orobtained in recombinant form. The hyaluronidase obtained by bacterialfermentation or recombinant techniques is preferred, such ashyaluronidase from Streptomyces koganeiensis ATCC 31394, in itsrecombinant form produced according to WO2014203133 (rHyal_Sk). Saidhyaluronidase is highly stable and suitable for the processes requiredfor covalent conjugation to nanoparticles. The presence of hyaluronidasein group X gives the system a hydrolytic capacity specific forhyaluronic acid.

Substituents: Groups Y and Z

They represent the variable part of the system, as they differ accordingto the intended applications of the nanoparticle system. According tothe invention, “active molecules” means both small molecules and complexmolecular structures (vaccines, antibodies, etc.). In general, group Yrepresents the active molecules, which may be actual medicaments ofnatural derivation (such as some 5-fluorouracil antitumorals,gemcitabine, doxorubicin, etc.), semi-synthetic, synthetic orrecombinant medicaments, diagnostic agents, radioactive agents,antibodies, camel nano-antibodies, biologically active substances suchas enzymes (e.g. human superoxide dismutase, native and/or modifiedmicrobial collagenase), proteins, peptides, hormones, growth factors,coagulation factors, cytokines, dyes, fluorophores (such as Cy3, Cy5,Cy7, fluorescein, rhodamine, naphthofluorescein, etc.), and in generalany active substance which can be conjugated to a covalent bond. Group Zmay be hydrogen (—H), an alkyl chain, a polyethylene glycol chain,sulphonic groups, directional molecules (antibodies, peptide chains,nucleic acids, polynucleotides, sense and antisense oligonucleotides,molecular conjugates containing RNA or DNA, water-soluble RNA, DNAvectors, natural, synthetic or recombinant vaccines) and other specificligands (once again defined as “active molecules”) to give thenanosystem selective properties.

Groups Y and Z can be variously combined with one another, depending onthe effect to be given to the nanoparticle system, its intended purposeand its use.

Various functional groups and known methods can be used for the covalentconjugations, such as bonds mediated by glutaraldehyde molecules, activeesters, bonds via maleimide, disulphide groups and squaric acid. Thebond via diethyl squarate is particularly preferred for the bond withhyaluronidase.

Schematically, the preparation process of the nanoparticle systemaccording to the invention comprises:

1. functionalization of nanoparticles, preferably polystyrenenanoparticles, with suitable functional groups, especially primary ortertiary amino groups (—NH₂), carboxyl groups , epoxy groups, preferablyamino groups;

2. functionalization of the particles thus obtained with the suitablyselected heterocarbon chain which, due to its orthogonal groups,subsequently leads to precise, selective functionalizations; in this waythe nanoparticles are given two or three temporarily protected armswhich, after selective deprotection, are ready to receive the activemolecules;

3. functionalization of the nanoparticle with hyaluronidase, preferablythe recombinant type rHyal_Sk (group X), in a reaction medium with pHvalues ranging between 7 and 11, in particular pH=10, and through acovalent bond, preferably via diethyl squarate. At this pH value thereare significant yields for the purpose of industrial scale-up (FIG. 2).

4. functionalization of the nanoparticle with one (bifunctionalization)or two (trifunctionalization) suitably selected active molecules, toobtain the system according to the invention.

Points 3 and 4 of the process thus schematically illustrated can beinverted, according to the dimensions, the nature of the molecules andtheir stability under the process conditions.

A further object of the invention consists of pharmaceuticalcompositions comprising the nanoparticle systems thus obtained, mixedwith pharmaceutically acceptable excipients. The compositions accordingto the invention are typically in freeze-dried form for reconstitution,-aqueous suspension or gel, and may be given by subcutaneous,intradermal, intrathecal, intramuscular, intraperitoneal, intravenous,intra-arterial, transdermal, transcutaneous or transmucosaladministration or by inhalation, in particular by subcutaneous,intradermal, transcutaneous, transdermal, transmucosal or intramuscularadministration or by inhalation.

The invention is described in greater detail in the examples below.

EXAMPLE 1 Functionalization of Nanoparticles with Hyaluronidase rHyal_Sk

1. 1 mL of amino-functionalised polystyrene nanoparticles (solid content2% in water) was centrifuged at 13400 rpm for 6 minutes, the supernatantwas removed, and the nanoparticles were resuspended by sonication in 1mL of N,N-dimethylformamide (DMF peptide grade, Scharlab).

2. N-Fmoc-N″-succinyl-4,7,10-trioxa-1,13-tridecane-diamine (PEG spacer,Sigma Aldrich) (75 equivalents) in DMF was mixed with 75 oximeequivalents (VWR) for 4 minutes at room temperature with continuousstirring (1400 rpm). 75 equivalents of N,N′-diisopropylcarbodiimide(DIC, Fluorochem) were then added, and the resulting mixture was placedunder stirring (1400 rpm) for 2 minutes at room temperature.

3. This last solution (point 2) was added to the nanoparticles (point1), and the resulting mixture was left to react by sonication andstirring (1400 rpm) at 60° C. for 2 hours [FIG. 2 (i)].

4. The nanoparticles were then centrifuged, the supernatant was removed,and the nanoparticles were washed with DMF (1 mL×2). After the washesthe Fmoc group was removed with a 20% piperidine solution (SigmaAldrich) in DMF (peptide grade, Scharlab) (1 mL×2) [FIG. 2 (ii)].

5. After deprotection, the nanoparticles were centrifuged at 13400 rpmfor 6 minutes, the supernatant was removed and the nanoparticles wereresuspended by sonication in 1 mL of a 1% solution ofN,N-diisopropylethylamine (DIPEA, Sigma Aldrich) in absolute ethanol(Scharlab), water (1:1) (v/v). 75 equivalents of3,4-diethoxy-3-cyclobutene-1,2-dione (diethyl squarate, Sigma Aldrich)were then added, and left to react under stirring (1400 rpm) at 25° C.for 15 hours [FIG. 2 (iii)].

6. The nanoparticles obtained were then centrifuged, and washed withdeionised water after removal of the supernatant (1 mL×2).

The nanoparticles (point 6) were then centrifuged again, the supernatantwas removed and the nanoparticles were resuspended in a 50 mM solutionof sodium borate (1 mL) containing 100 μg of rHyal_Sk [FIG. 2 (iv)]. Thenanoparticles were dispersed by sonication and a 1N solution of NaOH wasadded to maintain an environment at pH 10.

The final suspension was left to react under stirring (1400 rpm) at 25°C. for a further 15 hours. The nanoparticles were then centrifuged, thesupernatant was removed and the nanoparticles were washed with: (1) aPBS 1×buffer at pH 7.4 (1 mL) followed by (2) a 1% solution of bovineserum albumin (BSA, Sigma) and 40 mM ethanolamine (Sigma) in PBS buffer(1 mL). The nanoparticles were preserved in a PBS 1×buffer at pH 7, at4° C. (1 mL).

EXAMPLE 2 Bifunctionalization of Nanoparticles with Hyaluronidase(rHyal_Sk) and a Fluorophore (Cy5)

Addition to the Nanoparticles of the Carbon ChainN-Fmoc-N″-Succinyl-4,7,10-Trioxa-1,13-Tridecane-Diamine (PEG Spacer,Sigma Aldrich)

1. 1 mL of functionalised amino nanoparticles (solid content 2% inwater), prepared as described above, was centrifuged at 13400 rpm for 6minutes, the supernatant was removed, and the nanoparticles wereresuspended by sonication in 1 mL of N,N-dimethylformamide (DMF peptidegrade, Scharlab).

2. N-Fmoc-N″-succinyl-4,7,10-trioxa-1,13-tridecan-diamine (PEG spacer,Sigma Aldrich) (75 equivalents) in DMF was mixed with 75 of oximeequivalents (VWR) for 4 minutes at room temperature with continuousstirring (1400 rpm). 75 equivalents of N,N′-diisopropylcarbodiimide(DIC, Fluorochem) were then added, and the resulting mixture was placedunder stirring (1400 rpm) for 2 minutes at room temperature.

3. The solution described in point 2 was thus added to the nanoparticles(point 1), and the resulting mixture was left to react by sonication andstirring (1400 rpm) at 60° C. for 2 hours [FIG. 4 (i)].

4. The nanoparticles were then centrifuged, and after removal of thesupernatant were washed with DMF (1 mL×2). After the washes the Fmocgroup was deprotected with a 20% solution of piperidine (Sigma Aldrich)in DMF (peptide grade, Scharlab) (1 mL×2) [FIG. 4 (ii)].

5. After deprotection, the nanoparticles were centrifuged, thesupernatant was removed and the nanoparticles were washed with DMF (1mL×2) and methanol (synthesis grade, Scharlab) (1 mL×2). Thenanoparticles were preserved in deionised water (1 mL) at 4° C.

Bifunctionalization with Fmoc and Dde Groups

6. 1 mL of nanoparticles with PEG spacer obtained as described in thepreceding point (solid content 2% in water) (point 5) was centrifuged at13400 rpm for 6 minutes, the supernatant was removed, and thenanoparticles were resuspended by sonication in 1 mL of DMF.

7. Fmoc-Lys (Dde)-OH (GLBiochem) (75 equivalents) in DMF was mixed with75 oxime equivalents (VWR) for 4 minutes at room temperature undercontinuous stirring (1400 rpm). 75 equivalents of DIC (Fluorochem) werethen added, and the resulting mixture was placed under stirring (1400rpm) for 2 minutes at room temperature.

8. This last solution (point 7) was added to the nanoparticles (point5), and the resulting mixture was left to react by sonication andstirring (1400 rpm) at 60° C. for 2 hours [FIG. 4 (iii)].

9. The nanoparticles were then centrifuged, and after removal of thesupernatant were washed with DMF (1 mL×2). After the washes the Fmocgroup was removed with a 20% solution of piperidine in anhydrous DMF (1mL×2).

10. A PEG spacer was then added in the position previously protected bythe Fmoc group, repeating the steps described in points 1-4 [FIG. 4(iv-v)].

Conjugation of Fluorophore Cy5 to Nanoparticles

11. 1 mL of nanoparticles bifunctionalised with Fmoc and Dde, preparedas described in points 1-10 (solid content 2% in water), was centrifugedat 13400 rpm for 6 minutes, the supernatant was removed and thenanoparticles were resuspended by sonication in 1 mL of DMF. The Ddegroup was then selectively deprotected by treatment with hydroxylaminehydrochloride (1.80 mmol) (Acros)/imidazole (1.35 mmols) (Acros) inNMP:DMF (5:1) and stirring (1400 rpm) at 25° C. for 1 hour. 75equivalents of sulpho-NHS ester Cy5 (Lumiprobe) were then added and leftto react under stirring (1400 rpm) at 25° C. for 15 hours in the dark[FIG. 4 (vi-vii)].

12. After the 15-hour reaction, the nanoparticles were centrifuged, thesupernatant was removed and the nanoparticles were washed with DMF (1ml×2). Finally, the nanoparticles were preserved in deionised water (1mL) at 4° C. in the dark.

Conjugation of rHyal_Sk to Nanoparticles

13. When the nanoparticles had been conjugated to Cy5, the Fmoc groupwas deprotected. The nanoparticles were centrifuged, and the supernatantremoved and washed with DMF (1 ml×2). After the washes the Fmoc groupwas removed with a 20% solution of piperidine in DMF (1 ml×2) [FIG. 4(viii)].

14. After deprotection, the nanoparticles were centrifuged at 13400 rpmfor 6 minutes, the supernatant was removed and the nanoparticles wereresuspended by sonication in 1 mL of a 1% solution ofN,N-diisopropylethylamine (DIPEA, Sigma Aldrich) in absolute ethanol(Scharlab), water (1:1) (v/v). 75 equivalents of3,4-diethoxy-3-cyclobutene-1,2-dione (diethyl squarate, Sigma Aldrich)were then added and left to react under stirring (1400 rpm) at 25° C.for 15 hours in the dark [FIG. 4 (ix)].

15. After the 15-hour reaction, the nanoparticles were centrifuged andwashed with deionised water after removal of the supernatant (1 mL).

16. The nanoparticles (point 15) were then centrifuged again, thesupernatant was removed and the nanoparticles were resuspended in a 50mM solution of sodium borate (1 mL) containing 100 μg of rHyal_Sk [FIG.4 (x)]. The nanoparticles were dispersed by sonication and a 1N solutionof NaOH was added to maintain an environment at pH 10. The finalsuspension was left to react under stirring (1400 rpm) at 25° C. for afurther 15 hours in the dark. After the 15-hour reaction, thenanoparticles were centrifuged, the supernatant was removed and thenanoparticles were washed with: (1) a PBS 1×buffer at pH 7.4 (1 mL)followed by (2) a 1% solution of bovine serum albumin (BSA, Sigma) and40 mM ethanolamine (Sigma) in PBS buffer (1 mL). Finally, thenanoparticles were preserved in a PBS 1×buffer at pH 7, at 4° C. (1 mL)in the dark.

An identical procedure can be used to bond fluorophore Cy7, an analogousresult being obtained.

EXAMPLE 3 Bifunctionalization of Nanoparticles with rHyal_Sk andCapecitabine

Addition of the Carbon ChainN-Fmoc-N″-Succinyl-4,7,10-Trioxa-1,13-Tridecane-Diamine to theNanoparticles

See Example 2, points 1-5

Bifunctionalization with Fmoc and Dde Groups

See Example 2, points 6-10

Conjugation of Capecitabine to Nanoparticles

11. 1 mL of polystyrene nanoparticles bifunctionalised with Fmoc andDde, prepared as described in points 1-10 (solid content 2% in water),was centrifuged at 13400 rpm for 6 minutes, the supernatant was removedand the nanoparticles were resuspended by sonication in 1 mL of DMF. TheDde group was then selectively deprotected by treatment withhydroxylamine hydrochloride (1.80 mmol)/imidazole (1.35 mmols) inNMP:DMF (5:1) and stirring (1400 rpm) at 25° C. for 1 hour [FIG. 4(vi)]. An equimolar solution of succinic anhydride (75 equivalents) andDIPEA (75 equivalents) was then added, and dispersed by sonication andstirring (1400 rpm) at 60° C. for 2 h. Finally, the nanoparticles werewashed with DMF (1 mL×2).

12. The nanoparticles thus obtained (succinic-NP) were activated byincubation with 200 μL of an equimolar solution of oxime (50equivalents) and DIC (50 equivalents) for 4 hours, at room temperature,stirring at 1400 rpm. Capecitabine (Sigma Aldrich) (1 equivalent, 1mg/100 L DMF) and DMAP (Sigma) (0.5 mL) were then added under stirring(1400 rpm) at room temperature for 12 hours [FIG. 4 (vii)]. After thereaction, the nanoparticles were centrifuged and washed with DMF (1mL×2) and methanol (1 mL×2) after removal of the supernatant. Thenanoparticles were preserved in deionised water (1 mL) at 4° C.

Conjugation of rHyal_Sk to Nanoparticles

13. When the nanoparticles had been conjugated to capecitabine, the Fmocgroup was deprotected. The nanoparticles were centrifuged and washedwith DMF (1 mL×2) after removal of the supernatant. After the washes theFmoc group was removed with a 20% solution of piperidine in anhydrousDMF (1 mL×2) [FIG. 4 (viii)].

14. After deprotection, the nanoparticles were centrifuged at 13400 rpmfor 6 minutes, the supernatant was thus removed and the nanoparticleswere resuspended by sonication in 1 mL of a 1% solution ofN,N-diisopropylethylamine (DIPEA) in ethanol:water (1:1) (v/v). 75equivalents of 3,4-diethoxy-3-cyclobutene-1,2-dione (diethyl squarate,Sigma Aldrich) were then added, and the resulting mixture was left toreact under stirring (1400 rpm) at 25° C. for 15 hours [FIG. 4 (ix)].

15. After the 15-hour reaction, the nanoparticles were centrifuged, thesupernatant was removed and the nanoparticles were washed with deionisedwater (1 mL).

16. The nanoparticles (point 15) were then centrifuged again, thesupernatant was removed and the nanoparticles were resuspended in a 50mM solution of sodium borate (1 mL) containing 100 μg of rHyal_Sk. Thenanoparticles were dispersed by sonication and a 1N solution of NaOH wasadded to maintain an environment at pH 10. The final suspension was thenleft to react under stirring (1400 rpm) at 25° C. for a further 15 hours[FIG. 4 (x)]. After the 15-hour reaction the nanoparticles werecentrifuged, the supernatant was removed and the nanoparticles werewashed with a PBS 1×buffer at pH 7.4 (1 mL) followed by a 1% solution ofbovine serum albumin (BSA) and 40 mM ethanolamine in PBS buffer (1 mL).The nanoparticles were preserved in a PBS 1×buffer at pH 7, at 4° C. (1mL).

EXAMPLE 3B Bifunctionalization of Nanoparticles with rHyal_Sk andDoxorubicin (Doxo-Hyaluspheres)

Addition of the Carbon ChainN-Fmoc-N″-Succinyl-4,7,10-Trioxa-1,13-Tridecane-Diamine to theNanoparticles

Diamine to the Nanoparticles

See Example 2, points 1-5

Bifunctionalization with Fmoc and Dde Groups

See Example 2, points 6-10

Conjugation of Doxorubicin to Nanoparticles

11. 1 mL of nanoparticles bifunctionalised with Fmoc and Dde, preparedas described in points 1-10 (solid content 2% in water), was centrifugedat 13400 rpm for 6 minutes, the supernatant was removed and thenanoparticles were resuspended by sonication in 1 mL of DMF. The Ddegroup was then selectively deprotected by treatment with hydroxylaminehydrochloride (1.80 mmol)/imidazole (1.35 mmols) in NMP:DMF (5:1) andstirring (1400 rpm) at 25° C. for 1 hour [FIG. 4 (vi)]. An equimolarsolution of succinic anhydride (75 equivalents) and DIPEA (75equivalents) was then added, and dispersed by sonication and stirring(1400 rpm) at 60° C. for 2 h. Finally, the nanoparticles were washedwith DMF (1 mL×2).

12. The nanoparticles thus obtained (succinic-NP) were activated byincubation with 200 μL of an equimolar solution of oxime (75equivalents) and DIC (75 equivalents) for 4 hours, at room temperature,stirring at 1400 rpm. The nanoparticles were then centrifuged, thesupernatant was removed and the nanoparticles were resuspended in asolution of hydrazine hydrate (55% solution in water), 75 equivalents in1 mL of DMF. The nanoparticles were dispersed by sonication and left toreact under stirring (1400 rpm) at 25° C. for 15 hours.

13. After the reaction the nanoparticles were washed with DMF, methanoland PBS at pH 7.4.

14. The nanoparticles (point 13) were then washed with PBS at pH 6 (3×1mL) and resuspended in a solution of doxorubicin (5 equivalents) in PBSat pH 6 (1 mL). The nanoparticles were then dispersed by sonication andleft to react under stirring (1000 rpm) at 50° C. for 15 hours.

15. After the reaction, the nanoparticles were centrifuged, and washedwith PBS pH 7.4 (1 mL×2). The nanoparticles were preserved in PBS pH 7.4(1 mL) at 4° C. [FIG. 4 (vii)]

Conjugation of rHyal_Sk to Nanoparticles

16. When the nanoparticles had been conjugated to doxorubicin, the Fmocgroup was deprotected. The nanoparticles were centrifuged and washedwith DMF (1 mL×2) after removal of the supernatant. After the washes theFmoc group was removed with a 20% solution of piperidine in anhydrousDMF (1 mL×2) [FIG. 4 (viii)].

17. After deprotection, the nanoparticles were centrifuged at 13400 rpmfor 6 minutes, the supernatant was thus removed and the nanoparticleswere resuspended by sonication in 1 mL of a 1% solution ofN,N-diisopropylethylamine (DIPEA) in ethanol:water (1:1) (v/v). 75equivalents of 3,4-diethoxy-3-cyclobutene-1,2-dione (diethyl squarate,Sigma Aldrich) were then added, and the resulting mixture was left toreact under stirring (1400 rpm) at 25° C. for 15 hours [FIG. 4 (ix)].

18. After the 15-hour reaction, the nanoparticles were centrifuged, thesupernatant was removed and the nanoparticles were washed with deionisedwater (1 mL).

19. The nanoparticles (point 18) were then centrifuged again, thesupernatant was removed and the nanoparticles were resuspended in a 50mM solution of sodium borate (1 mL) containing 100 μg of rHyal_Sk. Thenanoparticles were dispersed by sonication and a 1N solution of NaOH wasadded to maintain an environment at pH 10. The final suspension was thenleft to react under stirring (1400 rpm) at 25° C. for a further 15 hours[FIG. 4 (x)]. After the 15-hour reaction, the nanoparticles werecentrifuged, the supernatant was removed and the nanoparticles werewashed with a PBS 1×buffer at pH 7.4 (1 mL) followed by a 1% solution ofbovine serum albumin (BSA) and 40 mM ethanolamine in PBS buffer (1 mL).The nanoparticles were preserved in a PBS 1×buffer at pH 7, at 4° C. (1mL).

EXAMPLE 4 Bifunctionalization of Nanoparticles with rHyal_Sk and anAnti-EGF Receptor Antibody

Addition of Carbon ChainN-Fmoc-N-Succinyl-4,7,10-Trioxa-1,13-Tridecan-Diamine to theNanoparticles

See Example 2, points 1-5

Bifunctionalization with Fmoc and Dde Groups

See Example 2, points 6-10

Conjugation of rHyal_Sk to Nanoparticles

11. 1 mL of nanoparticles bifunctionalised with Fmoc and Dde, preparedas described in points 1-10 (solid content 2% in water), was centrifugedat 13400 rpm for 6 minutes, the supernatant was removed and thenanoparticles were resuspended by sonication in 1 mL of DMF. The Ddegroup was then selectively deprotected by treatment with hydroxylaminehydrochloride (1.80 mmol)/imidazole (1.35 mmols) in NMP:DMF (5:1) andstirring (1400 rpm) at 25° C. for 1 hour [FIG. 5 (vi)]. Thenanoparticles were then centrifuged at 13400 rpm for 6 minutes, thesupernatant was removed and the nanoparticles were resuspended bysonication in 1 mL of a 1% solution of N,N-diisopropylethylamine (DIPEA)in ethanol:water (1:1) (v/v). 75 equivalents of3,4-diethoxy-3-cyclobutene-1,2-dione (diethyl squarate, Sigma Aldrich)were then added and left to react under stirring (1400 rpm) at 25° C.for 15 hours [FIG. 5 (vii)].

12. After the 15-hour reaction, the nanoparticles were centrifuged andwashed with water (1 mL) after removal of the supernatant.

13. The nanoparticles (point 12) were then centrifuged again, thesupernatant was removed and the nanoparticles were resuspended in a 50mM solution of sodium borate (1 mL) containing 100 μg of rHyal_Sk [FIG.5 (viii)]. The nanoparticles were dispersed by sonication and a 1Nsolution of NaOH was added to maintain an environment at pH 10. Thefinal suspension was then left to react under stirring (1400 rpm) at 25°C. for a further 15 hours.

14. After the 15-hour reaction, the nanoparticles were centrifuged, thesupernatant was removed, and the nanoparticles were washed with a PBS1×buffer at pH 7.4 (1 mL) and then with a 1% solution of bovine serumalbumin (BSA) and 40 mM ethanolamine in PBS buffer (1 mL). Finally, thenanoparticles were preserved in a PBS 1×buffer at pH 7, at 4° C. (1 mL).

Conjugation of Anti-EGF Receptor Antibody to Nanoparticles

15. When the nanoparticles had been conjugated to rHyal_Sk, the Fmocgroup was deprotected. The nanoparticles were centrifuged and washedwith DMF (1 mL×2) after removal of the supernatant. After the washes theFmoc group was removed with a 20% solution of piperidine in DMF (1mL×2).

16. 1 mL of nanoparticles (solid content 2% in water) was centrifuged at13400 rpm for 6 minutes, the supernatant was removed, and thenanoparticles were resuspended by sonication in 1 mL of DMF. Thefollowing solutions were then added: a) LC-SPDP (Thermo Scientific) 1mg/mL in DMF (dispersed by sonication), followed by (b) 20 equivalentsof DIPEA. The resulting mixture was left under stirring (1400 rpm) at25° C. for 12 hours. After 12 hours, a 1M solution of DTT was added, andthe resulting mixture was further incubated for 3 hours at 25° C. understirring (1400 rpm).

17. After the 3-hour reaction the nanoparticles were centrifuged, andwashed with water after removal of the supernatant (1 mL×2).

18. An activated SMCC antibody (Monoclonal Anti-EGF Receptor antibody,Sigma Aldrich) was then added at the concentration of (1.5 mg/mL) andincubated for 3 hours, under stirring (1400 rpm) at 25° C. [FIG. 5(ix)].

19. After the reaction with the antibody, the nanoparticles werecentrifuged, the supernatant was removed, and the nanoparticles werewashed with water (1 mL×2). The nanoparticles were preserved indeionised water (1 mL) at 4° C.

The resulting nanosystems were characterised for fluorescence byfluorescence microscopy and flow cytofluorometry (when they contained afluorophore), and for hyaluronidase activity, measured by turbidimetricassay in order to detect any variations in their intrinsic properties,as described in the following examples.

EXAMPLE 4B Bifunctionalization of Nanoparticles with rHyal_Sk and anAnti-EGF Receptor Antibody and Preparation for Trifunctionalization

Addition of Carbon ChainN-Fmoc-N-Succinyl-4,7,10-Trioxa-1,13-Tridecan-Diamine to theNanoparticles

See Example 2, points 1-5

Bifunctionalization with Fmoc and Dde Groups

See Example 2, points 6-10

Conjugation of (1R,8S,9S)—(1R,8S,9s)-Bicyclo[6.1.0]Non-4-yn-9-ylmethylN-Succinimidyl Carbonate (BCN-NHS) for Trifunctionalization

11. 1 mL of nanoparticles bifunctionalised with Fmoc and Dde, preparedas described in points 1-10 (solid content 2% in water), was centrifugedat 13400 rpm for 6 minutes, the supernatant was removed and thenanoparticles were resuspended by sonication in 1 mL of DMF. The Fmocgroup was then selectively deprotected by treatment with 20% piperidinein DMF (1 mL×2) [FIG. 5B (vi)].

12. The nanoparticles were washed with DMF (2 times) and dispersed bysonication, centrifuged at 13400 rpm for 6 minutes and the supernatantremoved.

13. A solution of BCN-NHS (Sigma Aldrich) (2 equivalents) and DIPEA (1equivalent) in DMF was prepared [FIG. 5B (vii)].

14. The solution (point 13) was added to the nanoparticles prepared inpoint 12, and the resulting mixture was sonicated and left to reactunder stirring at 1400 rpm, at 25° C., overnight [FIG. 5B (viii)].

15. After the reaction (point 14), the nanoparticles were centrifuged,the supernatant was removed and the nanoparticles were washed with DMF,methanol and water.

Conjugation of rHyal_Sk to Nanoparticles

16. 1 mL of nanoparticles bifunctionalised with BCN and Dde, prepared asdescribed in points 1-15 (solid content 2% in water), was centrifuged at13400 rpm for 6 minutes, the supernatant was removed, and thenanoparticles were resuspended by sonication in 1 mL of DMF. The Ddegroup was then selectively deprotected by treatment with hydroxylaminehydrochloride (1.80 mmol)/imidazole (1.35 mmols) in NMP:DMF (5:1) andstirring (1400 rpm) at 25° C. for 1 hour. The nanoparticles were thencentrifuged at 13400 rpm for 6 minutes, the supernatant was removed andthe nanoparticles were resuspended by sonication in 1 mL of a 1%solution of N,N-diisopropylethylamine (DIPEA) in ethanol:water (1:1)(v/v). 75 equivalents of 3,4-diethoxy-3-cyclobutene-1,2-dione (diethylsquarate, Sigma Aldrich) were then added, and left to react understirring (1400 rpm) at 25° C. for 15 hours [FIG. 5B (ix)].

17. After the 15-hour reaction, the nanoparticles were centrifuged andwashed with water (1 mL) after removal of the supernatant.

18. The nanoparticles (point 17) were then centrifuged, the supernatantwas removed and the nanoparticles were resuspended in a 50 mM solutionof sodium borate (1 mL) containing 100 μg of rHyal_Sk. The nanoparticleswere dispersed by sonication and a 1N solution of NaOH was added tomaintain an environment at pH 10. The final suspension was then left toreact under stirring (1400 rpm) at 25° C. for a further 15 hours [FIG.5B (x)].

19. After the 15-hour reaction, the nanoparticles were centrifuged, thesupernatant was removed, and the nanoparticles were washed with a PBS1×buffer at pH 7.4 (1 mL) and then with a 1% solution of bovine serumalbumin (BSA) and 40 mM ethanolamine in PBS buffer (1 mL). Finally, thenanoparticles were preserved in a PBS 1×buffer at pH 7, at 4° C. (1 mL).

Conjugation of Anti-EGF Receptor Antibody to Nanoparticles

20. The nanoparticles prepared in point 19 were centrifuged, and thesupernatant removed. At this point, a previously activated Ac-N₃solution (Monoclonal Anti-EGF Receptor antibody, Sigma Aldrich) wasadded. The mixture was incubated for 15 hours under stirring (1000 rpm)at 25° C.

21. After the reaction with the antibody, the nanoparticles werecentrifuged, the supernatant was removed, and the nanoparticles werewashed with PBS (1 mL×2). The nanoparticles were preserved in PBS (1 mL)at 4° C. [FIG. 5B (xi)].

The nanosystems obtained were characterised for coupling and antibodyspecificity by gel electrophoresis and flow cytofluorometry, and forhyaluronidase activity, which was measured by turbidimetric assay todetect any variations in their intrinsic properties, according to thefollowing examples.

EXAMPLE 4C Tri-Functionalization of Nanoparticles with a Fluorophore,rHyal_Sk and an Anti-EGF Receptor Antibody

Addition of the Carbon ChainN-Fmoc-N″-Succinyl-4,7,10-Trioxa-1,13-Tridecane-Diamine to theNanoparticles

See Example 2, points 1-5

Bifunctionalization with Fmoc and Dde Groups

See Example 2, points 6-10

Conjugation of (1R,8S,9S)-(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethylN-succinimidyl Carbonate (BCN-NHS) for Trifunctionalization

See Example 4B, points 11-15

16. 1 mL of polystyrene nanoparticles bifunctionalised with BCN and Dde,prepared as described in points 1-15 (solid content 2% in water), wascentrifuged at 13400 rpm for 6 minutes, the supernatant was removed andthe nanoparticles were resuspended by sonication in 1 mL of DMF. The Ddegroup was then selectively deprotected by treatment with hydroxylaminehydrochloride (1.80 mmol)/imidazole (1.35 mmols) in NMP:DMF (5:1) andstirring (1400 rpm) at 25° C. for 1 hour [FIG. 5C (ix)].

17. Fmoc-Lys (Dde)-OH (GLBiochem) (75 equivalents) in DMF was mixed with75 oxime equivalents (V WR) for 4 minutes at room temperature undercontinuous stirring (1400 rpm). 75 equivalents of DIC (Fluorochem) werethen added, and the resulting mixture was placed under stirring (1400rpm) for 2 minutes at room temperature.

18. This last solution (point 17) was added to the nanoparticles (point16), and left to react by sonication and stirring (1400 rpm) at 60° C.for 2 hours [FIG. 5C (x)].

Conjugation of Fluorophore Cy5 to Nanoparticles

19. 1 mL of nanoparticles trifunctionalised with Fmoc and Dde, preparedas described in points 1-18 (solid content 2% in water), was centrifugedat 13400 rpm for 6 minutes, the supernatant was removed and thenanoparticles were resuspended by sonication in 1 mL of DMF. The Ddegroup was then selectively deprotected by treatment with hydroxylaminehydrochloride (1.80 mmol) (Acros)/imidazole (1.35 mmols) (Acros) inNMP:DMF (5:1) and stirring (1400 rpm) at 25° C. for 1 hour. 75equivalents of sulpho-NHS ester Cy5 (Lumiprobe) were then added and leftto react under stirring (1400 rpm) at 25° C. for 15 hours in the dark[FIG. 5C (xi-xii)].

20. After the 15-hour reaction, the nanoparticles were centrifuged, thesupernatant was removed and the nanoparticles were washed with DMF (1ml×2). Finally, the nanoparticles were preserved in deionised water (1mL) at 4° C. in the dark.

Conjugation of rHyal_Sk to Nanoparticles

21. When the nanoparticles had been conjugated to Cy5, the Fmoc groupwas deprotected. The nanoparticles were centrifuged, the supernatant wasremoved and the nanoparticles were washed with DMF (1 ml×2). After thewashes the Fmoc group was deprotected with a 20% solution of piperidinein DMF (1 ml×2) [FIG. 5C (xiii)].

22. After deprotection, the nanoparticles were centrifuged at 13400 rpmfor 6 minutes, the supernatant was removed and the nanoparticles wereresuspended by sonication in 1 mL of a 1% solution ofN,N-diisopropylethylamine (DIPEA, Sigma Aldrich) in absolute ethanol(Scharlab) and water (1:1) (v/v). 75 equivalents of 3,4-diethoxy-3-cyclobutene-1,2-dione (diethyl squarate, Sigma Aldrich) werethen added and left to react under stirring (1400 rpm) at 25° C. for 15hours in the dark [FIG. 5C (xiv)].

23. After the 15-hour reaction, the nanoparticles were centrifuged, thesupernatant was removed and the nanoparticles were washed with deionisedwater (1 mL). The nanoparticles (point 22) were then centrifuged again,the supernatant was removed and the nanoparticles were resuspended in a50 mM solution of sodium borate (1 mI.) containing 100 μg of rHyal_Sk[FIG. 5C (xv)]. The nanoparticles were dispersed by sonication and a 1Nsolution of NaOH was added to maintain an environment at pH 10. Thefinal suspension was then left to react under stirring (1400 rpm) at 25°C. for a further 15 hours in the dark. After the 15-hour reaction, thenanoparticles were centrifuged, the supernatant was removed and thenanoparticles were washed with (1) a PBS 1×buffer at pH 7.4 (1 mL)followed by (2) a 1% solution of bovine serum albumin (BSA, Sigma) and40 mM ethanolamine (Sigma) in PBS buffer (1 mL). Finally, thenanoparticles were preserved in a PBS 1×buffer at pH 7, at 4° C. (1 mL).

Identical methods can be used to bond fluorophore Cy7, and a similarresult will be obtained.

Conjugation of Anti-EGF Receptor Antibody to Nanoparticles

24. The nanoparticles prepared in point 23 were centrifuged, and thesupernatant removed. At this point, a previously activated Ac-N₃solution (Monoclonal Anti-EGF Receptor antibody, Sigma Aldrich) wasadded. The mixture was incubated for 15 hours under stirring (1000 rpm)at 25° C. After the reaction with the antibody, the nanoparticles werecentrifuged, the supernatant was removed, and the nanoparticles werewashed with PBS (1 mL×2). The nanoparticles were preserved in PBS (1 mL)at 4° C. [FIG. 5 C (xvi)].

The nanosystems obtained were characterised for antibody coupling by gelelectrophoresis, and for antibody specificity and fluorescence(fluorophore coupling) by flow cytofluorometry. Hyaluronidase activitywas measured by turbidimetric assay to detect any variations in theirintrinsic properties, according to the following examples.

EXAMPLE 5 Evaluation of Enzyme Activity of rHyal_Sk AfterFunctionalization with Nanoparticles as Described in Example 1

The enzymatic activity of the nanosystem was measured by turbidimetricdetermination conducted on the excess supernatant recovered from thepreparation stages of Example 1, according to the following protocol.

Preparation of Reaction Buffers

1. Substrate Buffer (0.05% Solution of Hyaluronic Acid (w/v)).

Prepare an 0.5 mg/mL solution of hyaluronic acid in 300 mM of phosphatebuffer (pH 5.35). Heat the solution to 95° C. under stirring at 500 rpmfor 30 minutes (the time required to solubilise the hyaluronic acid).Then cool the solution to 37° C. and maintain it at 37° C. (until use).

Dilution Buffer (Enzyme).

Phosphate buffer (30 mM) containing 0.82% sodium chloride (pH 7.0, 37°C.)

2. Horse Serum Solution.

24 mM of sodium acetate and 79 mM of acetic acid containing 30% (v/v)horse serum (pH 3.75, 25° C.).

A. Preparation of standard curve.

The blank used is prepared with a mixture of 100 mL substrate buffer and100 μL enzyme dilution buffer (see points 1 and 2 above).

The master solution is prepared by adding 4 μL standard bovinehyaluronidase (1 unit per mL) to 196 μL enzyme dilution buffer. Six 1:1(v/v) serial dilutions are performed for the preparation of the curve.

B. Preparation of nanosystem suspension (nanoparticles covalentlyconjugated to the enzyme rHyal_sk). 10 μL of suspension is mixed with 90μl of enzyme dilution buffer and 100 ml of substrate buffer.

C. Incubate for 30 minutes at 37° C., stirring at 500 rpm.

D. Add 400 μL of horse serum solution (point 3).

E. Incubate for 30 minutes at 37° C., stirring at 500 rpm.

Measure the optical density at 640 nm.

The results of the turbidimetric analysis demonstrate that the enzymeretains hyaluronidase activity of around 97%. This confirms theefficiency of the system and demonstrates the high stability of rHyal_Skeven after the process of coupling to the nanoparticles.

EXAMPLE 6 Evaluation of Activity of the Nanosystem Obtained from Example2 (rHyal_Sk and Fluorophore Cy5)

The fluorescence and hyaluronidase activity of the nanosystem of Example2 were evaluated (FIG. 6A).

1 mL of nanosystem suspension was centrifuged and resuspended in 100 μLof water. 5 μL of the latter solution was then diluted in 100 μL for theflow cytometry studies. The flow cytometry was conducted with aFACSCanto II (Becton Dickinson & Co., N.J., USA) flow cytometer, usingan excitation laser at 633 nm. Unconjugated nanoparticles were used asnegative control. A Nikon A1R+/A1+ confocal laser microscope system wasused for the confocal microscopy analysis.http://www.nikoninstruments.com/Information-Center/Confocal Unconjugatednanoparticles were used as negative control.

The microscopy and cytofluorometry data demonstrate its high level offluorescence (FIG. 6B, C). Moreover, the turbidimetric data indicate ahigh level of hyaluronidase activity of the labelled nanosystems (FIG.7). It can be concluded from both types of data that the two ingredientscovalently coupled to the same nanoparticle retain their respectivefluorescence and enzyme activities intact. Similar findings are obtainedwith the nanosystem carrying rHyal_Sk and fluorophore Cy7.

EXAMPLE 7 Evaluation of Penetration of the Compounds Described inExample 2 in Cell Models In Vitro

3 different models were used to simulate subcutaneous absorption of thenanosystems.

Model 1: Penetration of Cell Monolayers

Nanoparticles prepared as described in Example 2 (Cy5-HyaluSpheres) weretested in this study, and nanoparticles conjugated to fluorophore Cy5only (Cy5-FluoroSpheres) were used as control. Cells that play a crucialpart in the subcutaneous absorption processes, namely fibroblasts andendothelial cells, were used. Fibroblasts are the most numerous cells inthe connective tissue properly so called. Their function is to producethe fibers and macromolecular constituents of extracellular matrix(including hyaluronic acid). The endothelial cells constitute the cellmonolayer that lines the blood vessels and acts as a selective barrierto the passage of molecules from the tissues to the blood.

These studies were conducted in monolayer cell models to determine moredirectly the ability of each cell line used to capture theCy5-HyaluSphere nanoparticles. HUVEC endothelial cells and humanforeskin fibroblasts (HFF) were used, and absorption was evaluated byflow cytometry and fluorescence microscopy by displaying fluorophoreCy5.

10⁵ cells per well were plated for 24 hours in a 24-well plate with 500μL of RPMI to which 10% foetal bovine serum and antibiotics were added.The cells were left to adhere for 24 h in 5% CO2 at 37° C. The culturemedium was then aspirated and replaced with fresh medium containing thefollowing concentrations of Cy5-HyaluSphere nanoparticles: 1:10, 1:100,1:1000 and 1:10000 nanoparticles per cell. The exact number ofnanoparticles per cell was calculated with a spectrophotometric method(J.D. Unciti-Broceta, et al. 2015, Scientific Reports 5, 10091).Cy5-FluoroSpheres were used as controls for the study.

Using a 1:1000 concentration, it was observed that about 100% of HUVECcells internalize Cy5-HyaluSpheres [FIG. 8A]. In the case of HFF, whenthe concentration is 1:1000, no internalization of Cy5-HyaluSpherenanoparticles is observed [FIG. 8B]. When the concentration ofnanoparticles per cell was increased to 1:10000, it was observed thatonly 35% of HFF cells internalized the Cy5-HyaluSphere nanoparticles[FIG. 8B]. These results are particularly significant, because theydemonstrate that Cy5-HyaluSpheres: 1) are not easily internalized byfibroblasts, and can therefore cross the connective tissue; 2) penetratethe endothelial cells and are easily conveyed into the bloodstream.

Model 2: Cell Penetration in Three-Dimensional (3D) Models

Nanoparticles prepared as described in Example 2 (Cy5-HyaluSpheres) weretested in this study, and nanoparticles conjugated to Cy5 only(Cy5-FluoroSpheres) were used as control. Three-dimensional (3D) cellmodels that simulate the subcutaneous tissue environment were used. AMatrigel gel (Matrigel® Basement Membrane Matrix, Corning) was used forthe study. The composition of said matrix is characterised by thepresence of proteins, such as laminin, collagen IV, heparan sulphateproteoglycans, entactin/nidogen glycoprotein, and various growthfactors, such as epidermal growth factor, insulin-like growth factor,fibroblast growth factors, tissue plasminogen activators and othergrowth factors. A concentration of hyaluronic acid was added to theMatrigel for this study. The number of nanoparticles added to theMatrigel was 10000 per cell.

Briefly, a monolayer of endothelial cells (HUVEC) with a concentrationof 5000 cells per well was seeded on a monolayer of a type B gelatinsolution (SIGMA). The Matrigel was mixed at the ratio of 1:2 with theculture medium, and hyaluronic acid was added at the concentration of 3mg/mL. When the Matrigel had become semisolid, the Cy5-FluoroSpherenanoparticles (control) and Cy5-HyaluSpheres with a concentration of10000 nanoparticles per cell were added on the semisolid layer ofMatrigel, and incubated for 24 h (in different wells). Theinternalization efficiency of the Cy5-HyaluSpheres was evaluated byconfocal microscopy, evaluating the difference in cell absorption of thenanoparticles after crossing the Matrigel component; only theCy5-HyaluSpheres cross the Matrigel and are therefore internalized bythe HUVEC cells, unlike the controls, which are unable to cross theMatrigel layer, and are therefore not internalized by the HUVEC cells.

Model 3: Cell Penetration in Transwell Models

To evaluate the ability of the nanoparticles, prepared as described inExample 2 (Cy5-HyaluSpheres), to cross the endodermal layer, twoexperiments were conducted on Transwell® cell culture inserts (Corning).These are permeable support devices which are easy to use to create anenvironment for cell cultures very similar to the in vivo state.

Briefly, a thick layer of HUVEC endothelial cells was seeded on theinsert with 90% confluence to simulate the endothelial layer wherein thecells are closely connected. Subsequently, a layer of Matrigel, mixedwith 3 mg/mL of hyaluronic acid, was placed on the cell layer (50000cells per well). Cy5-HyaluSpheres and Cy5-FluoroSpheres were added onthe Matrigel layer and incubated for 72 hours. After the incubationperiod, the nanoparticles in the lower part of the well (under theinsert) were collected, and the number of nanoparticles recovered wasmeasured by flow cytometry. The results demonstrate that the presence ofhyaluronidase enables the Cy5-HyaluSpheres to cross the Matrigel layermore easily than the Cy5-FluoroSpheres (FIG. 9A).

In the second experiment, two layers of HUVEC cells were seeded in theinsert (over and under it) to test the ability of the Cy5-HyaluSpheresto be internalized by the endothelial cells (under the insert) aftercrossing the Matrigel layer and the first cell layer [FIG. 9B]. A layerof Matrigel, mixed with 3 mg/mL of hyaluronic acid, was placed on thefirst layer of HUVEC. Cy5-HyaluSpheres and Cy5-FluoroSpheres were addedon the Matrigel layer and incubated for 72 hours. Confocal miscroscopyanalysis (FIG. 9C) demonstrated that the Cy5-HyaluSpheres wereeffectively internalized by the HUVEC cells seeded under the insertafter crossing the Matrigel layer with hyaluronic acid and the firstcell monolayer.

EXAMPLE 8 Evaluation of Activity of Nanosystem Prepared as Described inExample 3B

This study evaluated the activity of the drug Doxorubicin andhyaluronidase, both bonded to the nanosystems according to the presentinvention and prepared as described in Example 3B (hereinafter calledDoxo-Hyalusphere).

The cytotoxic activity of Doxo-Hyalusphere was tested on tumour celllines 4T1 (breast cancer) and A549 (lung cancer), using the in vitro MTTassay.

For the purpose of the test, cell line 4T1 was cultured in RPMI medium(Gibco) and A549 in DMEM (Gibco). 10% FBS (foetal bovine serum, Gibco),1% L-glutamine (Gibco) and 1% penicillin/streptomycin (Gibco) were addedto both culture media at the temperature of 37° C. in the presence of 5%CO2.

The cells were plated in 24-well microplates (Nunc) (50000 cells perwell in 500 μl of medium). After 12 hours, 100 μl of culture mediumcontaining the various conditions studied was added to each well:

1) Nanoparticles with PEG (PEG-NP);

2) Nanoparticles with rHyal_Sk (Hyal-NP);

3) Doxorubicin in solution (Free DOX);

4) Doxorubicin bonded to nanoparticles (DOX-NP)

5) Doxo-HyaluSpheres (DOX-Hyal-NP).

The nanoparticle concentrations were established at 40000 per cell, andthe plates were incubated for 24 and 96 hours. The species containingdoxorubicin were prepared at a concentration equivalent to 3 nM of drug(1.6 ng/mL). The wells were washed with PBS (Phosphate Buffered Saline),and 100 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide [(MTT) (0.5 mg/ml in colourless medium)] was then added. Theplates were left to incubate at 37° C. in the presence of 5% CO2 for 4hours. The MTT was then removed, and 100 μL of a solution of Triton X100, isopropanol and 37% HCl (12M) was added.

Metabolic activity was evaluated after 30 minutes by reading the opticaldensity (OD) with an ELISA Reader (Bio-Rad) spectrophotometer at thewavelength of 570 nm. Each sample was prepared in triplicate, in threeindependent experiments. The fraction of viable cells was determined onthe basis of the ratio between the mean optical density of the treatedsamples and that of the untreated control sample.

As shown by the graphs in FIGS. 10A and B, tumour cells 4T1 treated withthe nanoparticles alone (PEG-NP) and with the nanoparticles conjugatedto rHyal_Sk and without medicament (Hyal-NP) do not exhibit anytoxicity, maintaining cell viability at around 100% (using cells treatedwith saline solution as controls). Treatment of the 4T1cell lines withthe free drug (Free-DOX, FIG. 10A-B) did not lead to cell death;however, when the same quantity of drug of the Free-DOX model is bondedto the nanosystem, its ability to kill 4T1 considerably increases, inthe case of both Dox-NP and DOX-Hyal-NP, which acquire considerablecytotoxic activity. After 24 hours' treatment tumour cell survival is34%, and this rate falls to 10% after 96 hours' treatment.Quantitatively identical results were obtained with a quantity of freedoxorubicin amounting to 0.75 μg/mL (FIGS. 10A, B, C), i.e. with a farhigher dose of medicament. This means that the systems according to theinvention are exceptionally efficient and enable doxorubicin to be usedat far lower concentrations than those currently known. Similar resultswere obtained with tumour line A549 (FIG. 11).

The enzymatic activity of the resulting nanosystem was measured byturbidimetric determination conducted according to the protocol reportedin Example 5. 2×10⁹ nanoparticles, corresponding to the activity of 1 Uof hyaluronidase, were used for the study. The results demonstrate thatthe enzyme retains 97.5% hyaluronidase activity. This confirms theefficiency of the system and demonstrates the high stability ofrHyal_Sk, even after the coupling process used to prepare thenanosystems described herein.

It is therefore obvious that the two constituents covalently bonded tothe same nanoparticle maintain their respective anti-tumoral activityand enzymatic activity intact.

EXAMPLE 9 Evaluation of Activity of Nanosystem Obtained as Described inExample 4B

A nanosystem containing rHyal_Sk and Anti-EGF receptor antibody(Ab-HyaluSpheres) was prepared as described in Example 4B.

The enzymatic activity of the nanosystem was measured by turbidimetricdetermination conducted according to the protocol reported in Example 5.These results demonstrate that the enzyme retains a hyaluronidaseactivity of around 98.8±2.5%. This confirms the high stability ofrHyal_Sk after the coupling process used to prepare the nanosystemsobtained as described in Example 4B.

Conjugation to the anti-EGFR monoclonal antibody was validated with anagarose gel electrophoresis test (Lee J. et al, 2010, BioconjugateChem., 21, 940-946).

Briefly, 10 μL of sample [(8 uL of nanosystems (Example 4) and 2uL of 5×DNA Loading Buffer Blue (Bioline)] are loaded onto 0.8% agarose gel andimmersed in a buffer consisting of 89 mM tris, 89 mM borate and 2 mMEDTA, pH 8.0 (TBE). The samples are run for 30 minutes under a voltageof 70 mV. The gel was stained according to standard protocols withCoomassie Blue at the end of the gel electrophoresis run. Thecolocalization of the nanosystems stained with Coomassie Blue indicatescoupling of the nanosystems with the anti-EGFR monoclonal antibody.

The method is based on the non-motility of the nanosystems in agarosegel. The nanosystems are unable to move from the wells, whereas the freeantibodies migrate towards the negative electrode because they have aslight positive charge. The efficiency of conjugation was thereforeevaluated by analyzing the preservation of the antibody in the gel well.FIG. 12 shows the gel obtained, reading of which demonstrates effectiveconjugation of Ab to the nanoparticles (lines 3-5, FIG. 12). The twocontrols were line 1: Ab in solution without coupling to thenanoparticles; line 2: nanoparticles without Ab.

EXAMPLE 10 Evaluation of Activity of Nanosystem Obtained as Described inExample 4C

A nanosystem containing a trifunctionalization with a fluorophore,rHyal_Sk and an anti-EGF receptor antibody (Cy5-Ab-HyaluSpheres) wasprepared as described in Example 4C.

The enzymatic activity of the nanosystem was measured by turbidimetricdetermination conducted according to the protocol reported in Example 5.These results demonstrate that the enzyme retains a hyaluronidaseactivity of around 98.8%. This confirms the efficiency of the system anddemonstrates the high stability of rHyal_Sk, even after thetrifunctionalization process obtained as described in Example 4C.

The efficiency of conjugation of the antibody to the nanosystem wasevaluated by agarose gel electrophoresis and staining with CoomassieBlue, as described in Example 9. FIG. 13 shows the results obtained:line 1) (control): free Ab; Line 2 (control) only corresponds to thenanoparticles; Lines 3-5: different concentrations of Ab conjugated tothe nanoparticles. The most intense bands were observed in wells 3, 4and 5, which correspond to a gradient of increasing concentrations. Thebands were quantified by imaging analysis using ImageJ software(National Institutes of Health).

The coupling of the fluorophore and the selectivity of theAntiEGFR-Cy5-HyaluSpheres was studied by monitoring their cellabsorption with flow cytometry. The study was conducted by comparing theinternalization results of the AntiEGFR-Cy5-HyaluSphere nanoparticles ontumour cell lines A549 and H520. Cell line A549 presents over-expressionof the EGFR receptor, and line H520 presents low expression of the EGFRreceptor. FIG. 14 shows the flow cytometry results. TheAntiEGFR-Cy5-HyaluSpheres exhibited (as expected) a greater ability topenetrate the A549 cells with over-expression of EGFR receptor (about99%) and a lower ability to penetrate the line with low expression ofthe receptor (about 22%). This demonstrates that the antibodyselectively guides the penetration of the nanoparticles towards the EGFRantigen (FIGS. 14B and C). The Cy5-HyaluSpheres nanoparticles were usedas control. Examples of possible fields of application of the inventioninclude:

a) Immunostaining (in vitro): the nanosystems are conjugated tofluorophores and specific directional antibodies and with hyaluronidase,which increases their permeabilization when added to cell cultures. Theantibodies guide the nanosystems to molecular recognition, bonding tothe antigen or to target regions containing the antigen.

In this way said regions are highlighted by the fluorophores;

b) Single particle-tracking (in vivo): nanosystems conjugated tofluorophores and binders specific for membrane receptors (antibodies)bond to them outside the cell and make them visible. The hyaluronidaseallows them to enter the bloodstream by means of subcutaneousadministration. The movements and trajectories of the receptor can bemonitored by displaying the labelled nanosystems.

c) Targeted pharmacological treatment, specifically antitumoraltreatment: in this case the nanosystem transports an antitumoral drug.If the recognition of the tumour cells takes place via membranereceptors, antibodies can be used to ensure that the nanosystem ispreferably directed to sick cells wherein the receptors areover-expressed. The systems according to the invention are particularlysuitable for tumors characterised by an accumulation of hyaluronic acidin the tissues. Tumors that produce an abnormal accumulation ofhyaluronic acid which, by compressing the surrounding vessels, preventsor at least slows the transport of the drugs administered to the targetsite, particularly affect organs such as the breast, pancreas, colon andprostate. The excess hyaluronic acid accumulated also provides thetumour with an excellent growth substrate. In such situations it isessential to have a system that delivers the drug and simultaneouslyenables it to act completely, by eliminating a major mechanical andbiological obstacle. When tumour cells are recognised, the hyaluronidaseincreases their permeabilization by destroying the layers of hyaluronicacid that protect the tumour environment. After crossing the coating ofhyaluronic acid, the nanosystems release the antitumoral medicament,which thus acts more effectively. In this type of application, thenanosystem acts as directional carrier, giving the antitumoralmedicament a selective action. Moreover, the presence of hyaluronidasecan allow subcutaneous administration.

The nanosystems described herein therefore present the advantages ofbeing inert, biocompatible, and easily synthesized and functionalisedwith any type of molecule. They also retain intact the characteristicsof the molecules bonded to them and, in the case of some molecules,allow alternatives to the conventional administration routes to be used.

The nanosystems according to the invention therefore representmultipotent, flexible, reproducible, easily scalable systems, which canbe used as molecular transporters for diagnostic, prognostic andtherapeutic purposes.

1. Nanoparticle system comprising polymeric nanoparticles consisting ofa natural, synthetic or semisynthetic polymer or copolymer, bonded to aheterocarbon chain by covalent bonds between the functional groupspresent on the polymer or copolymer and the functional groups present onsaid heterocarbon chain, a hyaluronidase and one or more activemolecules covalently bonded to said heterocarbon chain.
 2. Nanoparticlesystem as claimed in claim 1 wherein the synthetic or natural polymersor copolymers are selected from polystyrene, polylactic acid, polylacticcoglycolic acid, poly(N-vinylpyrrolidone), polyethylene glycol,polycaprolactone, polyacrylic acid, polymethyl methacrylate,polyacrylamide, chitosan, gelatin, sodium alginate and albumin,preferably polystyrene.
 3. Nanoparticle system according to claim 1wherein the functional groups present on the polymer are selected fromprimary or tertiary amines, epoxides and carboxyls, preferably primaryor tertiary amines.
 4. Nanoparticle system according to claim 1 whereinthe nanoparticles have a size ranging from 50 nm to 2000 nm, preferablyfrom 100 to 500 nm, most preferably of 200 nm.
 5. Nanoparticle systemaccording to claim 1 wherein the hyaluronidase enzyme is of human,animal, vertebrate, bacterial or recombinant origin, preferablyrecombinant.
 6. Nanoparticle system according to claim 1 wherein theheterocarbon chain is selected from saturated carbon chains with 6 to 24carbons, methoxypolyethylene glycol chains, dimethylsuberimidate chains,polyethylene glycol chains, preferablyN-Fmoc-N-succinyl-4,7,10-trioxa-1,13-tridecanediamine (PEG spacer). 7.Nanoparticle system according to claim 1 wherein the functional groupsof the heterocarbon chain are selected from glutaraldehyde, maleimide,active esters, disulphide groups, squaric acid and derivatives thereof.8. Nanoparticle system according to claim 1, wherein the activemolecules are of natural, semisynthetic, synthetic or recombinantorigin.
 9. Nanoparticle system according to claim 7 wherein the activemolecules are selected from medicaments, diagnostic agents, radioactiveagents, antibodies, enzymes, proteins, peptides, hormones, growthfactors, coagulation factors, cytokines, dyes, fluorophores, antibodies,nucleic acids, polynucleotides, sense and antisense oligonucleotides,molecular conjugates containing RNA or DNA, soluble RNA, DNA vectors,and natural, synthetic or recombinant vaccines.
 10. Nanoparticle systemaccording to claim 8 wherein the enzymes are selected from humansuperoxide dismutase (hMnSOD) and native and/or modified bacterialcollagenase.
 11. Pharmaceutical compositions comprising the nanoparticlesystems of claim 1 in admixture with pharmaceutically acceptableexcipients.
 12. Pharmaceutical compositions according to claim 10 inlyophilized form for reconstitution, or in aqueous suspension or gelform.
 13. Pharmaceutical compositions according to claim 11 for thesubcutaneous, intradermal, intrathecal, intramuscular, intraperitoneal,intravenous, intra-arterial, transdermal, transcutaneous, transmucosaland inhalatory administration, preferably by subcutaneous, intradermal,transcutaneous, transdermal, transmucosal or intramuscularadministration or by inhalation.
 14. Nanoparticle system according toclaim 2 wherein the functional groups present on the polymer areselected from primary or tertiary amines, epoxides and carboxyls,preferably primary or tertiary amines.
 15. Nanoparticle system accordingto claim 2 wherein the nanoparticles have a size ranging from 50 nm to2000 nm, preferably from 100 to 500 nm, most preferably of 200 nm. 16.Nanoparticle system according to claim 3 wherein the nanoparticles havea size ranging from 50 nm to 2000 nm, preferably from 100 to 500 nm,most preferably of 200 nm.
 17. Pharmaceutical compositions comprisingthe nanoparticle systems of claim 2 in admixture with pharmaceuticallyacceptable excipients.
 18. Pharmaceutical compositions comprising thenanoparticle systems of claim 3 in admixture with pharmaceuticallyacceptable excipients.
 19. Pharmaceutical compositions comprising thenanoparticle systems of claim 4 in admixture with pharmaceuticallyacceptable excipients.
 20. Pharmaceutical compositions comprising thenanoparticle systems of claim 5 in admixture with pharmaceuticallyacceptable excipients.